The long-term goal of this proposal is to elucidate the mechanism of telomerase-mediated telomere replication. Telomeres are DNA-protein structures essential for the stability of chromosomes. Normally, telomeric DNA is lost as human somatic cells divide, eventually leading to chromosome instability and cell death or growth arrest whereas most tumors overcome the proliferative blockade imposed by telomere shortening via the activation of the reverse transcriptase telomerase. Activation of this enzyme is critical to convert normal cells to a tumorigenic state whereas inhibition of telomerase can lead to tumor regression. Telomerase therefore plays a fundamental role in cancer, and hence it is important to understand how this enzyme is regulated. One step in regulating telomerase is the assembly of an active enzyme complex at the telomeres. Indeed, in humans, mutations to the catalytic subunit of the enzyme appear to cripple the ability of telomerase to reach telomeres, and elongate telomeric DNA. While mutants of the enzyme provide a means to identify the proteins involved in assembling an active complex at telomeres, there are also two know proteins that may play a role in telomere recruitment: hPot1, the human homolog of the telomere-binding protein Cdc13p from budding yeast, which is known to recruit telomerase to telomere via interaction with other proteins, and PinX1, a human protein that binds directly to telomerase and telomeric proteins. Capitalizing on the cloning of these two proteins and our previous work of identifying mutants of telomerase, the following three aims are proposed to dissect the regulation of telomerase in human cells: Aim #1: Identify proteins that interact with the domain of telomerase implicated in telomere association. Aim #2: Characterize the role of hPot 1 in telomere stability and replication. Aim #3: Characterize the role of PinX1 and its novel associated protein PXE in telomerase-mediated telomere elongation. The accomplishment of the above aims will further our comprehension of how telomerase is regulated. Such information will be invaluable in elucidating the role of telomerase in cancer, which in turn could lay the foundation for the development of agents to inhibit telomerase function, ultimately for therapeutic use cancer treatment.